CN103773733B - A kind of Mammals intramuscular fat cell and Skeletal Muscle Cell layering co-culture method - Google Patents

Abstract

The invention discloses a kind of Mammals intramuscular fat cell and Skeletal Muscle Cell layering co-culture method.Take to butcher rear animal back longissimus muscle tissue, rinse and shred rear NTx enzymic digestion, postdigestive mixed liquid sieves; Collect filtrate, centrifugal, collect upper strata mature fat cell liquid and lower floor myocyte respectively, add appropriate serum free medium dilution washing respectively, centrifugal; Get upper strata centrifugate and lower floor's myocyte's liquid adds 25cm ²in culturing bottle, at 37 DEG C, 5%CO ₂cultivate in incubator.Mature fat cell due to buoyancy young pathbreaker floating adherent to upper strata " top ceiling " face, and myocyte sinks to the growth of culturing bottle bottom surface.This cell culture model can by the intramuscular fat cell of same for animal muscle tissue and myocyte layering Dual culture in same nutrient solution, thus provides new cell in vitro research model for the interaction of research mature fat cell secretory product on the impact of muscle cells differentiate or myocyte and adipocyte.

Description

A kind of Mammals intramuscular fat cell and Skeletal Muscle Cell layering co-culture method

Technical field

The invention belongs to technical field of cell biology, relate to a kind of Mammals intramuscular fat cell and Skeletal Muscle Cell layering co-culture method.

Background technology

The ratio of animal carcasses muscle and fatty tissue and structure and distribution thereof are the principal elements determining meat animals production performance and meat.In general muscle tissue forms primarily of Skeletal Muscle Cell.Fatty tissue can be divided into subcutaneous lipids, interior fat and intramuscular fat again, and it is ripe that different sites fatty deposits depends on the propagation of Preadipocyte, differentiation and polyester.Wherein intramuscular fat is useful fat, and be the basic substance forming marble grain meat, having participated in the formation of Meat Tenderness, succulence and Meat Flavor directly, is the important factor affecting meat flavor.In past 20 years, Chinese scholars utilizes clone such as 3T3-L1 pre-adipose cell lines and different animals such as the subcutaneous lipidss such as mouse, people, pig are studied Adipocyte Differentiation process on a cellular level and have achieved certain achievement.But because intramuscular fat is a small amount of tissue be mixed in muscle tissue, be separated with muscle tissue with difficulty, quantity such as to lack at the reason and be seldom separated and study.About Skeletal Muscle Cell research then main with Mouse Muscle clone C2C12 for experiment material, the separation of large-scale mammal Skeletal Muscle Cell is also relative less with research.

Along with to the continuous research of animal development rule and understanding, it is found that in muscle in animal body and its tangible animal development process of fat and be in a dynamic balance state.Preadipocyte In Vitro can be divided into ripe fat, and ripe fat also can dedifferente as precursor fatty even can to muscle cell transdifferentiation; Equally, under the condition that some is special, Muscle progenitor cells also can be divided into adipocyte.In addition, the cytokine of adipocyte secretion can affect the growth of myocyte by paracrine and Autocrine, and the muscle cells secrete factor also affecting lipocyte.Therefore, adipocyte and myocyte's single culture can only be observed respective independent events, and the interaction between them cannot be studied.

More than set forth and can draw, compare with subcutaneous lipids, the more worth research of molecular events of intramuscular fat cell, this just needs the intramuscular fat cell model that a purity is high, meanwhile, wants to understand the relation between intramuscular fat cell and myocyte, then need to cultivate same under same environmental conditions for two kinds of cells, the cytokine making it secrete interacts, like this, and just can better simulated animal internal milieu.So the independent and Dual culture in same nutrient solution by the intramuscular fat cell of same for animal muscle tissue and Skeletal Muscle Cell of the present invention's innovation, both can analyze separately Adipocyte Differentiation and dedifferente event, or myocyte's developmental mechanism, the interaction between two kinds of cells can be studied again, in addition method simply can operate, culture device requires low, is a kind of well cell culture model, has certain research promotion and be worth.

Summary of the invention

The object of the invention is to solve the problems such as Mammals intramuscular Preadipocyte In Vitro difficulty is separated, heteroproteose cell is many, solve a difficult problem for two kinds of cell layering independences and Dual culture simultaneously, a kind of Mammals intramuscular fat cell and Skeletal Muscle Cell layering co-culture method being provided, providing cell model for studying intercellular interaction.

A kind of Mammals intramuscular fat cell and Skeletal Muscle Cell layering co-culture method, comprise the following steps:

1) get animal back longissimus muscle, the muscle sample tissue that intramuscular fat is abundant, removing visible blood vessel and reticular tissue, use PBS wash buffer;

2) be aseptically cut into small pieces by tissue, adding concentration is the digestion of 1g/L NTx enzymic digestion liquid;

3), after digestion terminates, using in the DMEM/F12 substratum equal-volume containing 10% foetal calf serum and Digestive system, is the nylon screen filtration cell suspension of 250 μm with aperture;

4) step 3) filtrate through the centrifugal 8 ~ 15min of 900 ~ 1200r/m, get centrifugal after top layer suspension mature fat cell liquid add 25cm ²in culturing bottle, at 37 DEG C, 5%CO ₂quiescent culture in incubator; Described 25cm ²culturing bottle need fill the DMEM/F12 substratum containing 10% foetal calf serum in advance, at 37 DEG C, 5%CO ₂stationary incubation 4 ~ 6 hours in incubator, makes to keep in balance in culturing bottle;

5) get the centrifugal lower confluent monolayer cells of previous step and add erythrocyte cracked liquid washing, blow and beat gently with splitting erythrocyte, the centrifugal 3 ~ 8min of 900 ~ 1200r/m after effect 3 ~ 8min; Outwell upper strata centrifugate, add the lower confluent monolayer cells of serum-free DMEM/F12 substratum dilution washing, then be the nylon screen filtration of 80 μm with aperture, collect filtrate, the centrifugal 5min of 1000r/min; Outwell upper strata centrifugate, add the DMEM/F12 substratum of 500 μ L containing 10% foetal calf serum, being inoculated in the diameter be equipped with containing the DMEM/F12 substratum of 10% foetal calf serum after piping and druming cell dispersion is in 3.5cm culture dish, puts containing 37 DEG C, 5%CO ₂quiescent culture in incubator;

6) after inoculation 2 ~ 3 hours, take out Tissue Culture Dish, blow and beat the bottom of culture dish gently, sucking-off, containing the nutrient solution of myocyte, was transferred to step and is entered 4) in 25cm ²in culturing bottle, at 37 DEG C, 5%CO ₂culturing cell in incubator; The principle of this step is that Preadipocyte is adherent faster than Skeletal Muscle Cell, so Preadipocyte can be adherent at culture dish bottom grown after 2 ~ 3 hours, can not get off without trysinization, and blow and beat the bottom of culture dish gently, almost only containing Skeletal Muscle Cell in the nutrient solution sucked out;

7) mature fat cell due to buoyancy young pathbreaker floating adherent to " top ceiling " face, and Skeletal Muscle Cell can sink to a bottle bottom growth, thus just can realize after 24 hours in same culturing bottle, obtain the Skeletal Muscle Cell from the layering Dual culture of the same tissue of same animal and intramuscular fat cell.

Mammals intramuscular fat cell of the present invention and Skeletal Muscle Cell layering co-culture method, preferably include following steps:

1) get animal back longissimus muscle, the muscle sample tissue that intramuscular fat is abundant, cut off visible blood vessel and reticular tissue, with PBS wash buffer three times;

2) aseptically tissue is cut into 1mm ³the fritter of left and right, adds 1g/L NTx enzymic digestion liquid 37 DEG C, vibration digestion 90min in water-bath;

3), after digestion terminates, with containing in 10% foetal calf serum DMEM/F12 substratum equal-volume and Digestive system, be the nylon screen filtration cell suspension of 250 μm with aperture;

4) the centrifugal 10min of 1000r/m, gets 500 μ L4 × 10 ⁵top layer suspension mature fat cell liquid adds 25cm ²in culturing bottle, at 37 DEG C, 5%CO ₂quiescent culture in incubator; Described 25cm ²culturing bottle need fill the DMEM/F12 substratum containing 10% foetal calf serum in advance, at 37 DEG C, 5%CO ₂stationary incubation 4 ~ 6 hours in incubator, makes to keep in balance in culturing bottle;

5) outwell the upper strata centrifugate that previous step is centrifugal, take off confluent monolayer cells and add erythrocyte cracked liquid washing, piping and druming effect 3 ~ 5min is with splitting erythrocyte gently, the centrifugal 5min of 1000r/min;

6) outwell upper strata centrifugate, add the lower confluent monolayer cells of appropriate serum free medium dilution washing, then be the nylon screen filtration of 80 μm with aperture, collect filtrate, the centrifugal 5min of 1000r/min;

7) outwell upper strata centrifugate, add the DMEM/F12 substratum of 500 μ L containing 10% foetal calf serum, being inoculated in diameter after piping and druming cell dispersion is in 3.5cm culture dish, puts containing 37 DEG C, 5%CO ₂quiescent culture in incubator;

8) after inoculation 2 ~ 3 hours, take out Tissue Culture Dish, blow and beat the bottom of culture dish gently, sucking-off, containing the nutrient solution of myocyte, was transferred to step and is entered 4) in 25cm ²in culturing bottle, at 37 DEG C, 5%CO ₂culturing cell in incubator;

9) mature fat cell due to buoyancy young pathbreaker floating adherent to " top ceiling " face, and Skeletal Muscle Cell can sink to a bottle bottom growth, thus just can realize after 24 hours in same culturing bottle, obtain the Skeletal Muscle Cell from the layering Dual culture of the same tissue of same animal and intramuscular fat cell.

Described concentration is 1g/L NTx enzymic digestion liquid for adding 20g/L bovine serum albumin and 1g/L Collagenase I type obtains in serum-free DMEM nutrient solution.

The Mammals intramuscular fat cell of the layering Dual culture obtained according to the method described in the present invention and Skeletal Muscle Cell.

Beneficial effect:

The present invention is compared with existing cell culture technology, and its advantage shows:

(1) the present invention compares collagenase digestion single culture Skeletal Muscle Cell and single culture Preadipocyte In Vitro, and step is less, technology is simpler, can disposable acquisition two kinds of cells.

(2) the present invention and independent cultivate myocyte and independently cultivate Preadipocyte In Vitro and compare, can save test and spend half.

(3) the present invention utilizes the feature that mature fat cell buoyancy is little, and what obtain more than 99% purity dedifferentes Preadipocyte In Vitro, solves the problems such as Mammals intramuscular Preadipocyte In Vitro difficulty is separated, cell purity is not high.

(4) the invention solves the technical barrier of the same tissue of same animal two kinds of cells (adipocyte and myocyte) layering independence and Dual culture.

(5) to survive efficiency high for cultured cells of the present invention, through repeatedly observing adipocyte that confirmation cultivates in this way and Skeletal Muscle Cell vigor is good, cell characteristic is obvious, consistent with two kinds of independent cultured cells forms, and all express respective marker gene.Simultaneously, when can be used for studying co-cultivation, different cell-secretion factor is on the change of cytodifferentiation, growth, genetic expression and impact, it is the cell model done mutually between a kind of good research adipocyte and myocyte, compensate for external can not simulated animal body fat tissue and the interactional test defect of muscle tissue very well, for exploring mammal fat further and MB developmental mechanism provides useful test materials.

Accompanying drawing explanation

Fig. 1 mature fat cell floats to culturing bottle " top " adherent growth and dedifferentes gradually as Preadipocyte In Vitro

The intramuscular Preadipocyte In Vitro that Fig. 2 dedifferentes is divided into adipocyte again

Preadipocyte In Vitro in Fig. 3 differentiation expresses fatty marker gene

Note: 1,2,3,4 represent differentiation the 2nd day, the 4th day, the 6th day, the 8th day PPAR γ expression respectively; 5,6,7,8 represent differentiation the 2nd day, the 4th day, the 6th day, the 8th day C/EBP alpha expression situation respectively.

Fig. 4 Preadipocyte In Vitro marker gene Pref ?1 agarose gel electrophoresis result

Fig. 5 culturing bottle " bottom " Skeletal Muscle Cell form

The RT of Fig. 6 Skeletal Muscle Cell marker gene MyoD and Myf5 ?PCR electrophorogram

Note: 1,2 represent top adipocyte group and bottom inoblast group respectively.

Fig. 7 the inventive method schematic diagram.

Fig. 8 intramuscular Preadipocyte In Vitro and myocyte's cell total rna extract figure

Embodiment

Embodiment 1:

Meat depends primarily on muscle cell types and intramuscular fat content.3T3 ?the lipid substrate microtubule cell (SVF) of various animals of L1 Preadipocyte In Vitro system and original cuiture be the main cell model studying lipocyte proliferation, differentiation at present.But these cell models respectively have shortcoming: as 3T3 ?L1 clone derive from 17 ~ 19 day-old Mice embryonic cells, chromosome aneuploid, can not well reflect animal body environment; Although the SVF of original cuiture can simulated animal internal milieu preferably, this cell purity is not high, is unfavorable for scrutiny.In view of above problem, people are finding a kind of Preadipocyte In Vitro model that can reflect intramuscular fat cell differentiation procedure more accurately.And skeletal muscle accounts for 40% ?50% of the weight of animals, be important livestock product, that sets up Skeletal Muscle Cell model right solution muscle cell types is formed with vital effect.

Pig is one of topmost meat mammalian livestock in China, and the formation mechenism of its meat is the focus of numerous focus of attention always.In addition, the various physiological process of pig is more close with the mankind, and the ability of its accumulation fat is the strongest, is the model animal of a kind of good research human obesity and livestock and poultry body fat transition deposition.For this reason, we establish pig intramuscular fat cell and myocyte's stratified in vitro co-culture model with " top ceiling " culture method.

1 test materials

1.1 laboratory animal and tissue

Laboratory animal is that pig is delivered in fattening for sale, anosis through clinical examination health, butchers in rear 30min and gets longissimus dorsi muscle muscle.

1.2 instruments and apparatus

Lamina air flow filtration chamber, CO ₂cell culture incubator, inverted phase contrast microscope, Bechtop, electronic analytical balance, general refrigerator, automatic dual pure water distiller, full-automatic pressure steam sterilizer, DK ?8D electric heating constant temperature tank, generic centrifuge, beaker, graduated cylinder, spirit lamp, the curved tweezer of ophthalmology, stainless steel cell sieve and nylon screen, centrifuge tube, pipettor, cell counting count board, culture plate and culture dish etc.

1.3 reagent and preparation

The DMEM/F12 substratum of DMEM/F12 substratum, 10% foetal calf serum, NTx enzyme, foetal calf serum, TRIzol total RNA extraction reagent box, Reverse Transcription box and PCR reaction kit, OilRed ?O dye liquor etc.

NTx enzymic digestion liquid: serum-free DMEM/F12 substratum, 20g/L bovine serum albumin, 1g/L Collagenase I type (facing the used time adds, and uses after filtering).

Erythrocyte cracked liquid: 154mmol/LNH ₄cl, 10mmol/LKHCO ₃, 0.1mmol/LEDTA, tri-distilled water.

Oil red O dye liquor: 0.5g oil red O, adds the Virahol of 100mL98%, puts in 60 DEG C of thermostat containers and spends the night, and oil red O is fully dissolved.Face the used time to get 4mL distilled water and add 6mL stoste, filter after leaving standstill 30min and use.

The DMEM/F12 substratum that the present invention uses can be bought by commercially available approach, also can prepare voluntarily, for DMEM substratum and F12 substratum 1:1 mix.DMEM/F12 substratum containing 10% foetal calf serum is the foetal calf serum of interpolation 10% in DMEM/F12 substratum.

2 test methods

2.1 operating process

1) get animal back longissimus muscle, the muscle sample tissue that intramuscular fat is abundant, cut off visible blood vessel and reticular tissue, with PBS wash buffer three times;

2) aseptically tissue is cut into 1mm ³the fritter of left and right, adds NTx enzyme (1g/L) Digestive system digestion 90min (37 DEG C, vibrate in water-bath);

3), after digestion terminates, using in the DMEM/F12 substratum equal-volume containing 10% foetal calf serum and Digestive system, is the nylon screen filtration cell suspension of 250 μm with aperture;

4) the centrifugal 10min of 1000r/m, gets 500 μ L4 × 10 ⁵top layer suspension mature fat cell liquid adds 25cm ²in culturing bottle, at 37 DEG C, 5%CO ₂quiescent culture in incubator; Described 25cm ²culturing bottle need fill the DMEM/F12 substratum containing 10% foetal calf serum in advance, at 37 DEG C, 5%CO ₂stationary incubation 4 ~ 6 hours in incubator, makes to keep in balance in culturing bottle;

5) outwell the upper strata centrifugate that previous step is centrifugal, take off confluent monolayer cells and add erythrocyte cracked liquid washing, piping and druming effect 3 ~ 5min is with splitting erythrocyte gently, the centrifugal 5min of 1000r/min;

6) outwell upper strata centrifugate, add the lower confluent monolayer cells of appropriate serum-free DMEM/F12 substratum dilution washing, then be the nylon screen filtration of 80 μm with aperture, collect filtrate, the centrifugal 5min of 1000r/min;

7) outwell upper strata centrifugate, add the DMEM/F12 substratum of 500 μ L containing 10% foetal calf serum, the diameter being inoculated in the DMEM/F12 substratum that 10% foetal calf serum is housed after piping and druming cell dispersion is in 3.5cm culture dish, puts containing 37 DEG C, 5%CO ₂quiescent culture in incubator;

8) because the adherent speed of Preadipocyte is faster than Skeletal Muscle Cell, inoculate latter 3 hours, take out Tissue Culture Dish, blow and beat the bottom of culture dish gently, utilize the sucking-off of differential velocity adherent principle containing the nutrient solution of Skeletal Muscle Cell, transfer to step and enter 4) in 25cm ²in culturing bottle, at 37 DEG C, 5%CO ₂culturing cell in incubator;

9) mature fat cell due to buoyancy young pathbreaker floating adherent to " top ceiling " face, and Skeletal Muscle Cell can sink to a bottle bottom growth, just can realize after 24 hours in same culturing bottle, obtain the Skeletal Muscle Cell from the same tissue of same animal and intramuscular fat cell, and can layering Dual culture.The impact broken up Skeletal Muscle Cell for research intramuscular fat cell secreta like this or the interaction of Skeletal Muscle Cell and intramuscular fat cell supply a model.

2.2 morphological observation and qualification

2.2.1 morphological observation

Every day, observe culturing bottle " top " and " bottom " cell respectively, observation of cell growing state, paid special attention to the change of its form under inverted microscope, and record visible change feature and time of occurrence thereof, take pictures.

2.2.2 oil red O stain qualification intramuscular mature fat cell

PBS washed cell 2 times, 10% formaldehyde fixes 30min, and PBS washes 2 times, and oil red O stain 20min, PBS wash 1 time, and basis of microscopic observation lipid within endothelial cells takes on a red color.

2.2.3 intramuscular Preadipocyte In Vitro, Skeletal Muscle Cell qualification is dedifferented

Observation of cell, after mature fat cell dedifferentes completely, with RT ?round pcr detect Preadipocyte In Vitro and Skeletal Muscle Cell marker gene respectively, concrete operation step is as follows:

(1) intramuscular Preadipocyte In Vitro and myocyte's cell total rna extract, and step is shown in Fig. 8.

(2) cell total rna detects

The RNA extracted is dissolved in appropriate without in the DEPC water of RNAse.1% agarose gel electrophoresis, EB dye, observe under UV-light RNA integrity and with or without degraded and contaminating genomic DNA; With 500 times of DEPC water dilutions, measure wavelength 260,280 place OD value with ultraviolet spectrophotometer, calculate OD260/OD280 value and RNA concentration.Extract pollution-free, be 1.8 ~ 2.0 without degradation of rna OD260/OD280 value.If any DNA pollution, process with DNAse I.

(3) PCR primer

Application PrimerPremier5.0 software, according to the basic demand of design of primers, design adipocyte marker gene C/EBP α and PPAR γ, skeletal muscle marker gene MyoD and Myf5 ， β ?actin gene PCR amplimer, primer length 18 ~ 22bp.Synthesized by the raw work in Shanghai.

Table 1 adipocyte and myocyte marker gene RT ?PCR primer

(4) RT ?PCR system and reaction operate by product description.

3 results

3.1 intramuscular mature fat cell observationss

According to little and swim in this feature of upper strata of liquid containing significant quantities of fat, density in mature fat cell, be separated to mature fat cell by common centrifuging, and adopted " top ceiling " culture method culturing cell.Basis of microscopic observation floats to the top layer adherent growth (figure 1 ?A) of culturing bottle to mature fat cell.After oil red O stain, cell is positive (figure 1 ?B) completely; Adherent 72h inner cell is that single chamber fat drips mature fat cell form, and observes the adipocyte splitting into 2 cells; Occur after 72h that greasiness drips cell, start to present Fusoid cells form (figure 1 ?C); After 14d, in cell, fat drips completely dissolve, dedifferentes the Preadipocyte In Vitro (figure 1 ?D) into inoblast form

The 3.2 intramuscular Preadipocyte In Vitro of dedifferenting can be divided into adipocyte again

By in the intramuscular Preadipocyte In Vitro of dedifferenting (figure 2 ?the A) culture dish that goes down to posterity from culturing bottle independent cultivate 7 days after, find that the intramuscular Preadipocyte In Vitro of dedifferenting can be divided into the adipocyte (figure 2 ?B) dripped containing fat again.

The Preadipocyte In Vitro of 3.3 differentiation expresses fatty marker gene

The Preadipocyte In Vitro of dedifferenting express fat precursors marker gene Pref ?1, and do not express myocyte and indicate MyoD, as Fig. 3.In the intramuscular Preadipocyte In Vitro of dedifferenting in adipocyte again atomization, with RT ?PCR method detect the expression changing conditions of adipocyte marker gene PPAR γ and C/EBP α, result is 2 of cytodifferentiation, 4,6, within 8 days, all there is the expression of adipocyte marker gene PPAR γ and C/EBP α, as Fig. 4.

3.4 Skeletal Muscle Cells cultivations, observation and qualification result

Because the adherent speed of Skeletal Muscle Cell is slower than Preadipocyte, first according to " differential attachment method " principle, the cell of centrifugal lower floor is inoculated in culture dish and cultivates, after 3 hours, not adherent Skeletal Muscle Cell is sucked out, when adding the culturing bottle being full of substratum from culture dish; Feature less than mature fat cell according to Skeletal Muscle Cell buoyancy again, supposition can sink to adherent growth bottom culturing bottle.Result has into fibrous Growth of Cells (figure 5 ?A) bottom basis of microscopic observation to culturing bottle after 24 hours of incubation really, basis of microscopic observation when 4 days, discovery does not have heteroproteose cell purity very high (figure 5 ?B), and can to the muscle cells differentiate (figure 5 ?C) containing myotube shape

3.5 one-tenth fibrous cell expressing skeletal muscle marker gene

With " top " adipocyte for contrast, with RT ?PCR method detect myocyte marker gene MyoD and Myf5 expression, result " top " adipocyte does not express MyoD and Myf5, and bottom inoblast expresses MyoD and Myf5, illustrate that bottom inoblast is Skeletal Muscle Cell, as Fig. 6.

4 conclusions

Present method success obtains highly purified intramuscular mature fat cell and Skeletal Muscle Cell after 24 hours in same culturing bottle.Continuing to cultivate by 14 days, making intramuscular mature fat cell dedifferente fibrous cell's form of dripping for not containing fat completely.This cell expressing Preadipocyte In Vitro marker gene Pref ?1, and do not express other cells as skeletal muscle mark MyoD; This precursor fatty is cultured to converging state, goes down to posterity under being placed on adipogenic induction agent and cultivate, the cell oil red O stain of 99% is positive as a result; Adipocyte Differentiation key transcription factor PPAR γ and C/EBP α is expressed in atomization, with 3T3 ?the Preadipocyte In Vitro system atomization such as L1 consistent, illustrate that the Preadipocyte In Vitro of dedifferenting can be divided into mature fat cell again, prove Preadipocyte In Vitro; And the inoblast of lower floor expresses skeletal muscle marker gene MyoD and Myf5, and the cell of flesh tube-like condition can be formed, prove Skeletal Muscle Cell.

Further, these two kinds of cell deriveds clear (deriving from the longissimus dorsi muscle marble grain meat tissue of Mammals pig), can grow by syntrophism in same culture system.Frozen front and back cellular form and vigor good; Normally, heritability is good for chromosome number and caryogram; Without bacterium, virus and mycoplasma contamination.Prove it is feasible in this way, being a kind of high purity, high score rate, the Mammals intramuscular fat cell of low cost and myocyte's layering co-culture method, providing useful test materials for studying mammal meat Forming Mechanism further.

Claims (2)

1. Mammals intramuscular fat cell and a Skeletal Muscle Cell layering co-culture method, is characterized in that comprising the following steps:

1) get animal back longissimus muscle, the muscle sample tissue that intramuscular fat is abundant, removing visible blood vessel and reticular tissue, use PBS wash buffer;

2) be aseptically cut into small pieces by tissue, adding concentration is the digestion of 1g/L NTx enzymic digestion liquid;

3), after digestion terminates, using in the DMEM/F12 substratum equal-volume containing 10% foetal calf serum and Digestive system, is the nylon screen filtration cell suspension of 250 μm with aperture;

4) filtrate of step 3) is through the centrifugal 8 ~ 15min of 900 ~ 1200r/m, get centrifugal after top layer suspension mature fat cell liquid add 25cm ²in culturing bottle, at 37 DEG C, 5%CO ₂quiescent culture in incubator; Described 25cm ²culturing bottle need fill the DMEM/F12 substratum containing 10% foetal calf serum in advance, at 37 DEG C, 5%CO ₂stationary incubation 4 ~ 6 hours in incubator, makes to keep in balance in culturing bottle;

5) get the centrifugal lower confluent monolayer cells of previous step and add erythrocyte cracked liquid washing, blow and beat gently with splitting erythrocyte, the centrifugal 3 ~ 8min of 900 ~ 1200r/m after effect 3 ~ 8min; Outwell upper strata centrifugate, add the lower confluent monolayer cells of serum-free DMEM/F12 substratum dilution washing, then be the nylon screen filtration of 80 μm with aperture, collect filtrate, the centrifugal 5min of 1000r/min; Outwell upper strata centrifugate, add the DMEM/F12 substratum of 500 μ L containing 10% foetal calf serum, being inoculated in the diameter be equipped with containing the DMEM/F12 substratum of 10% foetal calf serum after piping and druming cell dispersion is in 3.5cm culture dish, puts containing 37 DEG C, 5%CO ₂quiescent culture in incubator;

6) after inoculation 2 ~ 3 hours, take out Tissue Culture Dish, blow and beat the bottom of culture dish gently, sucking-off, containing the nutrient solution of myocyte, was transferred to step and is entered 4) in 25cm ²in culturing bottle, at 37 DEG C, 5%CO ₂culturing cell in incubator;

7) mature fat cell due to buoyancy young pathbreaker floating adherent to " top ceiling " face, and Skeletal Muscle Cell can sink to a bottle bottom growth, thus just can realize after 24 hours in same culturing bottle, obtain the Skeletal Muscle Cell from the layering Dual culture of the same tissue of same animal and intramuscular fat cell;

Wherein, described concentration is 1g/L NTx enzymic digestion liquid for adding 20g/L bovine serum albumin and 1g/L Collagenase I type obtains in serum-free DMEM/F12 substratum.

2. Mammals intramuscular fat cell according to claim 1 and Skeletal Muscle Cell layering co-culture method, is characterized in that comprising the following steps:

1) get animal back longissimus muscle, the muscle sample tissue that intramuscular fat is abundant, cut off visible blood vessel and reticular tissue, with PBS wash buffer three times;

2) aseptically tissue is cut into 1mm ³the fritter of left and right, adds 1g/L NTx enzymic digestion liquid 37 DEG C, vibration digestion 90min in water-bath;

3), after digestion terminates, with containing in 10% foetal calf serum DMEM/F12 substratum equal-volume and Digestive system, be the nylon screen filtration cell suspension of 250 μm with aperture;

4) the centrifugal 10min of 1000r/m, gets 500 μ L4 × 10 ⁵top layer suspension mature fat cell liquid adds 25cm ²in culturing bottle, at 37 DEG C, 5%CO ₂quiescent culture in incubator; Described 25cm ²culturing bottle need fill the DMEM/F12 substratum containing 10% foetal calf serum in advance, at 37 DEG C, 5%CO ₂stationary incubation 4 ~ 6 hours in incubator, makes to keep in balance in culturing bottle;

5) outwell the upper strata centrifugate that previous step is centrifugal, take off confluent monolayer cells and add erythrocyte cracked liquid washing, piping and druming effect 3 ~ 5min is with splitting erythrocyte gently, the centrifugal 5min of 1000r/min;

6) outwell upper strata centrifugate, add the lower confluent monolayer cells of appropriate serum free medium dilution washing, then be the nylon screen filtration of 80 μm with aperture, collect filtrate, the centrifugal 5min of 1000r/min;

7) outwell upper strata centrifugate, add the DMEM/F12 substratum of 500 μ L containing 10% foetal calf serum, being inoculated in diameter after piping and druming cell dispersion is in 3.5cm culture dish, puts containing 37 DEG C, 5%CO ₂quiescent culture in incubator;

8) after inoculation 2 ~ 3 hours, take out Tissue Culture Dish, blow and beat the bottom of culture dish gently, sucking-off, containing the nutrient solution of myocyte, was transferred to step and is entered 4) in 25cm ²in culturing bottle, at 37 DEG C, 5%CO ₂culturing cell in incubator;

9) mature fat cell due to buoyancy young pathbreaker floating adherent to " top ceiling " face, and Skeletal Muscle Cell can sink to a bottle bottom growth, thus just can realize after 24 hours in same culturing bottle, obtain the Skeletal Muscle Cell from the layering Dual culture of the same tissue of same animal and intramuscular fat cell.